Contrasting secondary coordination sphere effects on spin density distribution in Red vs. Blue Cu azurin

Metalloproteins tune the electronic properties of metal active sites through a combination of primary and secondary coordination sphere effects (PCS and SCS) to efficiently perform an array of redox chemistry, including electron transfer (ET) and catalysis. A major influence of these effects is the anisotropic spatial distribution of redox-active molecular orbitals (RAMOs), which in turn dictates redox chemistry of the metalloproteins. While much progress has been made in understanding the SCS effects on RAMOs in individual native metalloproteins, it has been difficult to experimentally examine the influence of the same SCS effects on RAMOs with different spatial distributions. Taking advantage of our recent studies of SCS effect on blue copper azurin from Pseudomonas aeruginosa (Blue CuAz) and its M121H/H46E variant that closely mimic the red copper protein (Red CuAz), in which their RAMOs are dominated by either Cu-S_π or Cu-S_σ interactions, respectively, we herein compare and contrast how the same SCS modifications impact the electronic and geometric structures of blue and red Cu center in the same protein scaffold. Specifically, we expand our understanding of unpaired electron distribution at the Cu-binding site of Red CuAz and its SCS N47S, F114P, and F114N mutations using ¹H and ¹⁴N electron-nuclear double resonance (ENDOR) spectroscopy, and then further combine these data sets with recent studies and DFT calculations to provide insight into how these mutations differentially (or similarly) impact electronic structure in Red vs. Blue CuAz. We find that electrostatics produce similar effects in both Red and Blue CuAz, where the introduction of dipole moments in the vicinity of Cu and S produces changes in spin density distribution and of the same sign and comparable magnitude. However, disruption of H-bonding with S through the F114P mutation leads to opposing effects in Red vs. Blue CuAz, which we propose arise from differences in the conformation of Cys112 sidechain adapted in the absence the stabilizing S_C112⋯H-N backbone interaction.